In the field of a clinical test, analysis of hemocyte components in a sample is very useful for diagnosing a variety of diseases in a circulatory organ of a subject. Depending on a disease, the number of particular hemocytes is increased or decreased, or blood cells which are not usually present appear in peripheral blood in some cases.
In recent years, various automatic hemocyte counting devices, to which a principle of flow cytometry is applied, are commercially available. Using such devices, sorting and counting of hemocyte cells are performed in general laboratories. When these automatic hemocyte counting devices are used, sorting and counting of leukocytes in a sample can be automatically performed.
For sorting and counting leukocytes, first, erythrocytes in a blood sample are lyzed. When the resulting sample is guided to a detector, and an electric impedance signal is detected, leukocytes can be sorted into three kinds. Alternatively, by the following method, leukocytes can be sorted into five kinds; lymphocytes, monocytes, neutrophils, eosinophils, and basophils. First, erythrocytes in a blood sample are lyzed, and hemocytes in the resulting sample are stained with a fluorescent dye. Then, hemocytes are irradiated with excited light, and a fluorescent signal and a scattered light signal emitted from the hemocytes are detected. By analyzing those signals, leukocytes can be sorted into five kinds.
Since the number of basophils is usually small, only basophils may be measured without sorting leukocytes into five kinds by one measurement. Based on the property that basophils are hardly destroyed under acidic conditions as compared with other leukocytes, the number of basophils can be determined by treating a blood sample exclusively for measurement of basophils (see Japanese Examined Patent Publication (JP-B) No. 6-8817). When this result and the result of leukocyte sorting obtained by another method are combined, leukocytes can be sorted into five kinds more correctly.
Appearance of nucleated red blood cells (NRBC) often becomes a problem in leukocyte measurement. Since nucleated red blood cells have a nucleus, a nucleus remains even when erythrocytes are lyzing-treated. Since the remaining nucleus emits a signal similar to that of lymphocytes in the aforementioned measuring method, a plus error is generated at the time of measurement of the number of leukocytes. In order to exclude this influence, for example, there is a method of performing a treatment exclusive for measurement of nucleated red blood cells, and determining the number of nucleated red blood cells (Japanese Unexamined Patent Publication (JP-A) No. 10-339729), and subtracting the number of nucleated red blood cells from the number of leukocytes obtained by another method. By this method, the correct number of leukocytes can be obtained.
However, when a treatment exclusive for particular hemocytes is increased in order to correctly sort leukocytes, this is troublesome, and there is a possibility that a device is complicated, or scaled up. In addition, when a plurality of reagents exclusive for particular hemocytes are used, the cost of a total blood test is increased. From such a point of view, it is preferable that a treatment exclusive for particular hemocyte is performed as little as possible.
Measurement of basophils and nucleated red blood cells can be performed by treating a blood sample under acidic condition. Therefore, when a blood sample is treated under acidic condition, there is a possibility that both of basophils and nucleated red blood cells can be measured by one measurement. For example, JP-A No. 2002-148261 discloses a method of measuring basophils and erythroblasts (nucleated red blood cells) comprising mixing an aqueous solution containing an erythrocyte lyzing agent and a surfactant which bring leukocytes and abnormal cells into a state suitable for staining, with a sample, adding a dyeing solution containing a fluorescent dye to stain it, and measuring a fluorescent intensity and a scattered light intensity with a flow cytometer.
However, in the case of a specimen containing many leukocytes, the aforementioned conventional method does not sufficiently separate basophils and leukocytes other than basophils in some cases.